RESUMO
Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Animais , Cromatina/química , Cromossomos , Saccharomyces cerevisiae/genéticaRESUMO
As the second most prevalent hematologic malignancy, multiple myeloma (MM) remains incurable and relapses due to intrinsic or acquired drug resistance. Therefore, new therapeutic strategies that target molecular mechanisms responsible for drug resistance are attractive. Interactions of tumor cells with their surrounding microenvironment impact tumor initiation, progression and metastasis, as well as patient prognosis. This cross-talk is bidirectional. Tumor cells can also attract or activate tumor-associated stromal cells by releasing cytokines to facilitate their growth, invasion and metastasis. The effect of myeloma cells on bone marrow stromal cells (BMSCs) has not been well studied. In our study, we found that higher stiffness of BMSCs was not a unique characteristic of BMSCs from MM patients (M-BMSCs). BMSCs from MGUS (monoclonal gammopathy of undetermined significance) patients were also stiffer than the BMSCs from healthy volunteers (N-BMSCs). The stiffness of M-BMSCs was enhanced when cocultured with myeloma cells. In contrast, no changes were seen in myeloma cell-primed MGUS- and N-BMSCs. Interestingly, our data indicated that CD138⻠myeloma cells, but not CD138⺠cells, regulated M-BMSC stiffness. SDF-1 was highly expressed in the CD138⻠myeloma subpopulation compared with that in CD138⺠cells. Inhibition of SDF-1 using AMD3100 or knocking-down CXCR4 in M-BMSCs blocked CD138⻠myeloma cells-induced increase in M-BMSC stiffness, suggesting a crucial role of SDF-1/CXCR4. AKT inhibition attenuated SDF-1-induced increases in M-BMSC stiffness. These findings demonstrate, for the first time, CD138⻠myeloma cell-directed cross-talk with BMSCs and reveal that CD138⻠myeloma cells regulate M-BMSC stiffness through SDF-1/CXCR4/AKT signaling.
Assuntos
Quimiocina CXCL12/metabolismo , Células-Tronco Mesenquimais/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Sindecana-1/metabolismo , Fenômenos Biomecânicos , Ativação Enzimática/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Gamopatia Monoclonal de Significância Indeterminada/patologia , Cadeias Leves de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or "aptamers" because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4% aptamer content before selection to >15% aptamer content.
Assuntos
Aptâmeros de Nucleotídeos/química , Eletroforese Capilar/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Significance: Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth ( z ) information into a modification of the two-dimensional image. Physical insight into DOE designs will spur this expanding field. Aim: To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system. Approach: We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker. Results: Bead localization precision was < 25 nm in the transverse dimensions and ≤ 70 nm along the axial dimension over an axial range of 6 µ m . Higher axial precision ( ≤ 50 nm ) was achieved over a shallower focal depth of 2.9 µ m . 3D diffusion constants of chromatin matched expected values. Conclusions: Precise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.
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Imageamento Tridimensional , Dispositivos Ópticos , Imageamento Tridimensional/métodos , Microscopia , CromatinaRESUMO
The study of radiation effects on biological tissues is a diverse field of research with direct applications to improve human health, in particular in the contexts of radiation therapy and space exploration. Understanding the DNA damage response following radiation exposure, which is a key determinant for mutagenesis, requires reproducible methods for delivering known doses of ionizing radiation (IR) in a controlled environment. Multiple IR sources, including research X-ray and gamma-ray irradiators are routinely used in basic and translational research with cell and animal models. These systems are however not ideal when a high temporal resolution is needed, for example to study early DNA damage responses with live cell microscopy. Here, we characterize the dose rate and beam properties of a commercial, miniature, affordable, and versatile X-ray source (Mini-X). We describe how to use Mini-X on the stage of a fluorescence microscope to deliver high IR dose rates (up to 29 Gy/min) or lower dose rates (≤ 0.1 Gy/min) in live cell imaging experiments. This article provides a blueprint for radiation biology applications with high temporal resolution, with a step-by-step guide to implement a miniature X-ray system on an imaging platform, and the information needed to characterize the system.
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Microscopia , Radiobiologia , Animais , Radiação Ionizante , Raios XRESUMO
BACKGROUND: The epithelium forms a protective barrier against external biological, chemical and physical insults. So far, AFM-based, micro-mechanical measurements have only been performed on single cells and confluent cells, but not yet on cells in mature layers. METHODS: Using a combination of atomic force, fluorescence and confocal microscopy, we determined the changes in stiffness, morphology and actin distribution of human mammary epithelial cells (HMECs) as they transition from single cells to confluency to a mature layer. RESULTS: Single HMECs have a tall, round (planoconvex) morphology, have actin stress fibers at the base, have diffuse cortical actin, and have a stiffness of 1 kPa. Confluent HMECs start to become flatter, basal actin stress fibers start to disappear, and actin accumulates laterally where cells abut. Overall stiffness is still 1 kPa with two-fold higher stiffness in the abutting regions. As HMECs mature and form multilayered structures, cells on apical surfaces become flatter (apically more level), wider, and seven times stiffer (mean, 7 kPa) than single and confluent cells. The main drivers of these changes are actin filaments, as cells show strong actin accumulation in the regions where cells adjoin, and in the apical regions. CONCLUSIONS: HMECs stiffen, flatten and redistribute actin upon transiting from single cells to mature, confluent layers. GENERAL SIGNIFICANCE: Our findings advance the understanding of breast ductal morphogenesis and mechanical homeostasis.
Assuntos
Citoesqueleto de Actina/fisiologia , Células Epiteliais/citologia , Glândulas Mamárias Humanas/citologia , Organogênese , Células Cultivadas , Células Epiteliais/fisiologia , Feminino , Humanos , Glândulas Mamárias Humanas/fisiologia , Microscopia de Força AtômicaRESUMO
Particle tracking in living systems requires low light exposure and short exposure times to avoid phototoxicity and photobleaching and to fully capture particle motion with high-speed imaging. Low-excitation light comes at the expense of tracking accuracy. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure data sets, qualitatively improving the images. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic data sets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional data sets, whereas artifacts were introduced by the denoisers in three-dimensional data sets. Experimentally, we found that, while both supervised and unsupervised approaches improved tracking results compared with the original noisy images, supervised learning generally outperformed the unsupervised approach. We find that nicer-looking image sequences are not synonymous with more precise tracking results and highlight that deep learning algorithms can produce deceiving artifacts with extremely noisy images. Finally, we address the challenge of selecting parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optimal particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of this approach to critically evaluate artificial intelligence solutions for quantitative microscopy.
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Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Artefatos , Inteligência Artificial , Teorema de Bayes , Linhagem Celular Tumoral , Aprendizado Profundo , Humanos , Redes Neurais de Computação , Razão Sinal-RuídoRESUMO
Optogenetic control over neuronal firing has become an increasingly elegant method to dissect the microcircuitry of mammalian brains. To date, examination of these manipulations on neurotransmitter release has been minimal. Here we present the first in-depth analysis of optogenetic stimulation on dopamine neurotransmission in the dorsal striatum of urethane-anesthetized rats. By combining the tight spatial and temporal resolution of both optogenetics and fast-scan cyclic voltammetry we have determined the parameters necessary to control phasic dopamine release in the dorsal striatum of rats in vivo. The kinetics of optically induced dopamine release mirror established models of electrically evoked release, indicating that potential artifacts of electrical stimulation on ion channels and the dopamine transporter are negligible. Furthermore a lack of change in extracellular pH indicates that optical stimulation does not alter blood flow. Optical control over dopamine release is highly reproducible and flexible. We are able to repeatedly evoke concentrations of dopamine release as small as a single dopamine transient (50 nM). An inverted U-shaped frequency response curve exists with maximal stimulation inducing dopamine effluxes exceeding 500 nM. Taken together, these results have obvious implications for understanding the neurobiological basis of dopaminergic-based disorders and provide the framework to effectively manipulate dopamine patterns.
Assuntos
Corpo Estriado/metabolismo , Dopamina/genética , Dopamina/metabolismo , Estimulação Luminosa/métodos , Animais , Humanos , Masculino , Fenômenos Ópticos , Ratos , Ratos Sprague-DawleyRESUMO
The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab-on-Bead library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy-aided identification of single target-bound beads and the extraction--wet or dry--of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers.
Assuntos
Técnicas de Química Combinatória/métodos , Descoberta de Drogas/métodos , Ácidos Nucleicos Peptídicos/química , Sequência de Bases , Técnicas de Química Combinatória/instrumentação , Descoberta de Drogas/instrumentação , Corantes Fluorescentes/química , Biblioteca de PeptídeosRESUMO
Electrospun nanofibers manufactured from biocompatible materials are used in numerous bioengineering applications, such as tissue engineering, creating organoids or dressings, and drug delivery. In many of these applications, the morphological and mechanical properties of the single fiber affect their function. We used a combined atomic force microscope (AFM)/optical microscope technique to determine the mechanical properties of nanofibers that were electrospun from a 50:50 fibrinogen:PCL (poly-ε-caprolactone) blend. Both of these materials are widely available and biocompatible. Fibers were spun onto a striated substrate with 6 µm wide grooves, anchored with epoxy on the ridges and pulled with the AFM probe. The fibers showed significant strain softening, as the modulus decreased from an initial value of 1700 MPa (5-10% strain) to 110 MPa (>40% strain). Despite this extreme strain softening, these fibers were very extensible, with a breaking strain of 100%. The fibers exhibited high energy loss (up to 70%) and strains larger than 5% permanently deformed the fibers. These fibers displayed the stress-strain curves of a ductile material. We provide a comparison of the mechanical properties of these blended fibers with other electrospun and natural nanofibers. This work expands a growing library of mechanically characterized, electrospun materials for biomedical applications.
RESUMO
Despite many years of work on dopaminergic mechanisms of alcohol addiction, much of the evidence remains mostly correlative in nature. Fortunately, recent technological advances have provided the opportunity to explore the causal role of alterations in neurotransmission within circuits involved in addictive behaviors. Here, we address this critical gap in our knowledge by integrating an optogenetic approach and an operant alcohol self-administration paradigm to assess directly how accumbal dopamine (DA) release dynamics influences the appetitive (seeking) component of alcohol-drinking behavior. We show that appetitive reward-seeking behavior in rats trained to self-administer alcohol can be shaped causally by ventral tegmental area-nucleus accumbens (VTA-NAc) DA neurotransmission. Our findings reveal that phasic patterns of DA release within this circuit enhance a discrete measure of alcohol seeking, whereas tonic patterns of stimulation inhibit this behavior. Moreover, we provide mechanistic evidence that tonic-phasic interplay within the VTA-NAc DA circuit underlies these seemingly paradoxical effects.
RESUMO
The relationship between stress and alcohol-drinking behaviors has been intensively explored; however, neuronal substrates and neurotransmitter dynamics responsible for a causal link between these conditions are still unclear. Here, we optogenetically manipulated locus coeruleus (LC) norepinephrine (NE) activity by applying distinct stimulation protocols in order to explore how phasic and tonic NE release dynamics control alcohol-drinking behaviors. Our results clearly demonstrate contrasting behavioral consequences of LC-NE circuitry activation during low and high frequency stimulation. Specifically, applying tonic stimulation during a standard operant drinking session resulted in increased intake, while phasic stimulation decreased this measure. Furthermore, stimulation during extinction probe trials, when the lever press response was not reinforced, did not significantly alter alcohol-seeking behavior if a tonic pattern was applied. However, phasic stimulation substantially suppressed the number of lever presses, indicating decreased alcohol seeking under the same experimental condition. Given the well-established correlative link between stress and increased alcohol consumption, here we provide the first evidence that tonic LC-NE activity plays a causal role in stress-associated increases in drinking.
Assuntos
Locus Cerúleo , Neurônios , Comportamento de Ingestão de Líquido , NorepinefrinaRESUMO
Preventing cancer is vastly better than treating the disease in terms of a patient's quality of life and healthcare costs. Yet, to screen for chemopreventative drugs or evaluate interventions aimed at lowering cancer risk, quantitative readouts of risk are needed. In the breast and in other organs of epithelial origin, apical-basal polarity is key to homeostasis and is one of the first tissue characteristics lost during cancer initiation. Therefore, apical-basal polarity may be leveraged as an "architectural" determinant of cancer risk. A classic approach to quantify the localization of epithelial polarity markers is visual scoring at the microscope by trained investigators. This approach is time-intensive and limited to low throughput. To increase the speed, accuracy, and scoring volume, we developed an algorithm that essentially replaces the human eye to objectively quantify epithelial polarity in microscopy images of breast glandular units (acini). Acini in culture are identified based on a nuclear stain and the corresponding masks are divided into concentric terraces of equal width. This positional information is used to calculate radial intensity profiles (RP) of polarity markers. Profiles with a steep slope represent polarized structures, whereas more horizontal curves are indicative of non-polarized acini. To compare treatment effects, RP curves are integrated into summary values of polarity. We envision applications of this method for primary cancer prevention research with acini organoids, specifically (1) to screen for chemoprevention drugs, (2) for toxicological assessment of suspected carcinogens and pharmacological hit compounds, and (3) for personalized evaluation of cancer risk and risk-reducing interventions. The RadialProfiler algorithm developed for the MATLAB computing environment and for users without prior informatics knowledge is publicly available on the Open Science Framework (OSF).
RESUMO
Activity in the mesolimbic dopamine (DA) pathway is known to have a role in reward processing and related behaviors. The mesolimbic DA response to reward has been well-examined, while the response to aversive or negative stimuli has been studied to a lesser extent and produced inconclusive results. However, a brief increase in the DA concentration in terminals during nociceptive activation has become an established but not well-characterized phenomenon. Consequently, the interpretation of the significance of this neurochemical response is still elusive. The present study was designed to further explore these increases in subsecond DA dynamics triggered by negative stimuli using voltammetry in anesthetized rats. Our experiments revealed that repeated exposure to a tail pinch resulted in more efficacious DA release in rat nucleus accumbens. This fact may suggest a protective nature of immediate DA efflux. Furthermore, a sensitized DA response to a neutral stimulus, such as a touch, was discovered following several noxious pinches, while a touch applied before these pinches did not trigger DA release. Finally, it was found that the pinch-evoked DA efflux was significantly decreased by ethanol acutely administrated at an analgesic dose. Taken together, these results support the hypothesis that subsecond DA release in the nucleus accumbens may serve as an endogenous antinociceptive signal.
Assuntos
Dopamina/metabolismo , Etanol/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Estimulação Física/efeitos adversos , Animais , Estimulação Física/métodos , Ratos , Ratos Sprague-Dawley , Cauda/efeitos dos fármacos , Cauda/metabolismoRESUMO
Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation.
Assuntos
Neoplasias da Mama/patologia , Leptina/sangue , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Proteínas Adaptadoras de Transdução de Sinal , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Animais , Índice de Massa Corporal , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Leptina/genética , Leptina/metabolismo , Glândulas Mamárias Humanas/metabolismo , Camundongos Endogâmicos BALB C , Obesidade/metabolismo , Obesidade/patologia , Lesões Pré-Cancerosas , Fuso Acromático/metabolismo , Fuso Acromático/patologiaRESUMO
We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340 ± 30 nm, which simultaneously photoactivate a 7 × 7 matrix pattern of GFP-labeled histones, with spots 1.70 µm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.
Assuntos
Cromatina/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/metabolismo , Desenho de Equipamento , Histonas/química , Humanos , Iluminação , Microscopia de Fluorescência/instrumentaçãoRESUMO
Cancer treatment and treatment options are quite limited in circumstances such as when the tumor is inoperable, in brain cancers when the drugs cannot penetrate the blood-brain-barrier, or when there is no tumor-specific target for generation of effective therapeutic antibodies. Despite the fact that electromagnetic fields (EMF) in medicine have been used for therapeutic or diagnostic purposes, the use of non-ionizing EMF for cancer treatment is a new emerging concept. Here we summarize the history of EMF from the 1890's to the novel and new innovative methods that target and treat cancer by non-ionizing radiation.
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Campos Eletromagnéticos , Neoplasias/terapia , Animais , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/patologiaRESUMO
We develop a method, which utilizes a combined atomic force microscope (AFM)/fluorescence microscope and small copy number polymerase chain reaction (PCR), to affinity-select individual aptamer species in a single cycle from a small pool of random-sequence oligonucleotides (oligos). In this method, a library of small beads, each of which is functionalized with fluorescent oligos of different sequences, is created. This library of oligo-functionalized beads is flowed over immobilized target molecules on a glass cover slip. High-affinity target-specific aptamers bind tightly to the target for prolonged periods and resist subsequent washes, resulting in a strong fluorescence signal on the substrate surface. This signal is observed from underneath the sample via fluorescence microscopy. The AFM tip, situated above the sample, is then directed to the coordinates of the fluorescence signal and is used to capture a three-dimensional high-resolution image of the surface-bound bead and to extract the bead (plus attached oligo). The extracted oligo is PCR-amplified, sequenced, and may then be subjected to further biochemical analysis. Here, we describe the underlying principles of this method, the required microscopy instrumentation, and the results of proof-of-principle experiments. In these experiments, we selected aptamers in eight trials from a binary pool containing a 1:1 mixture of thrombin aptamer oligo and a nonsense oligo. In each of the eight trials, the positive control aptamer was successfully detected, imaged, extracted, and characterized by PCR amplification and sequencing. In no case was the nonsense oligo selected, indicating good selectivity at this early stage of technology development.
Assuntos
Aptâmeros de Nucleotídeos/química , Microscopia de Força Atômica/instrumentação , Microscopia de Fluorescência/métodos , Sequência de Bases , Microscopia de Força Atômica/métodos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
We have designed and tested a new, inexpensive, easy-to-make and easy-to-use calibration standard for atomic force microscopy (AFM) lateral force measurements. This new standard simply consists of a small glass fiber of known dimensions and Young's modulus, which is fixed at one end to a substrate and which can be bent laterally with the AFM tip at the other end. This standard has equal or less error than the commonly used method of using beam mechanics to determine a cantilever's lateral force constant. It is transferable, thus providing a universal tool for comparing the calibrations of different instruments. It does not require knowledge of the cantilever dimensions and composition or its tip height. This standard also allows direct conversion of the photodiode signal to force and, thus, circumvents the requirement for a sensor response (sensitivity) measurement.
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Algoritmos , Análise de Falha de Equipamento/métodos , Manometria/instrumentação , Microscopia de Força Atômica/instrumentação , Transdutores , Calibragem , Análise de Falha de Equipamento/normas , Manometria/métodos , Manometria/normas , Microscopia de Força Atômica/métodos , Microscopia de Força Atômica/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estados UnidosRESUMO
Blood clots, whose main structural component is a mesh of microscopic fibrin fibers, experience mechanical strain from blood flow, clot retraction and interactions with platelets and other cells. We developed a transparent, striated and highly stretchable substrate made from fugitive glue (a styrenic block copolymer) to investigate how mechanical strain affects lysis of single, suspended fibrin fibers. In this suspended fiber assay, lysis manifested itself by fiber elongation, thickening (disassembly), fraying and collapse. Stretching single fibrin fibers significantly hampered their lysis. This effect was seen in uncrosslinked and crosslinked fibers. Crosslinking (without stretching) also hampered single fiber lysis. Our data suggest that strain is a novel mechanosensitive factor that regulates blood clot dissolution (fibrinolysis) at the single fiber level. At the molecular level of single fibrin molecules, strain may distort, or hinder access to, plasmin cleavage sites and thereby hamper lysis. STATEMENT OF SIGNIFICANCE: Fibrin fibers are the major structural component of a blood clot. We developed a highly stretchable substrate made from fugitive glue and a suspended fibrin fiber lysis assay to investigate the effect of stretching on single fibrin fibers lysis. The key findings from our experiments are: 1) Fibers thicken and elongate upon lysis; 2) stretching strongly reduces lysis; 3) this effect is more pronounced for uncrosslinked fibers; and 4) stretching fibers has a similar effect on reducing lysis as crosslinking fibers. At the molecular level, strain may distort plasmin cleavage sites, or restrict access to those sites. Our results suggest that strain may be a novel mechanobiological factor that regulates fibrinolysis.